@article{li_he_la hovary_zhu_dong_liu_xing_liu_jie_ma_et al._2022, title={A de novo regulation design shows an effectiveness in altering plant secondary metabolism}, volume={37}, ISSN={["2090-1224"]}, url={http://dx.doi.org/10.1016/j.jare.2021.06.017}, DOI={10.1016/j.jare.2021.06.017}, abstractNote={Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.}, journal={JOURNAL OF ADVANCED RESEARCH}, publisher={Elsevier BV}, author={Li, Mingzhuo and He, Xianzhi and La Hovary, Christophe and Zhu, Yue and Dong, Yilun and Liu, Shibiao and Xing, Hucheng and Liu, Yajun and Jie, Yucheng and Ma, Dongming and et al.}, year={2022}, month={Mar}, pages={43–60} }
 @article{wu_la hovary_chen_li_eng_vallejo_qu_dewey_2020, title={An Efficient Stevia rebaudiana Transformation System and In vitro Enzyme Assays Reveal Novel Insights into UGT76G1 Function}, volume={10}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-020-60776-y}, abstractNote={Abstract Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacterium-mediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of UGT76G1 . By comparing the SG profiles among independent transgenic events, we demonstrated that altering UGT76G1 expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in E . coli , we show that two closely related UGT76G1 isoforms differ in their substrate specificities, providing new insights into mechanisms underlying the diversity of SG profiles that are observed across Stevia germplasm. Finally, we found evidence suggesting that alternative and/or aberrant splicing may serve to influence the ability of the plant to produce functional UGT76G1 transcripts, and possibly produce enzyme variants within the plant.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Wu, Qian and La Hovary, Christophe and Chen, Han-Yi and Li, Xu and Eng, Hayde and Vallejo, Veronica and Qu, Rongda and Dewey, Ralph E.}, year={2020}, month={Feb} }
 @article{ma_gandra_manoharlal_la hovary_xie_2019, title={Untargeted Metabolomics of Nicotiana tabacum Grown in United States and India Characterizes the Association of Plant Metabolomes With Natural Climate and Geography}, volume={10}, ISSN={1664-462X}, url={http://dx.doi.org/10.3389/fpls.2019.01370}, DOI={10.3389/fpls.2019.01370}, abstractNote={Climate change and geography affect all the living organisms. To date, the effects of climate and geographical factors on plant metabolome largely remain open for worldwide and local investigations. In this study, we designed field experiments with tobacco (Nicotiana tabacum) in India versus USA and used untargeted metabolomics to understand the association of two weather factors and two different continental locations with respect to tobacco metabolism. Field research stations in Oxford, North Carolina, USA, and Rajahmundry, Andhra Pradesh India were selected to grow a commercial tobacco genotype (K326) for 2 years. Plant growth, field management, and leaf curing followed protocols standardized for tobacco cultivation. Gas chromatography–mass spectrometry based unbiased profiling annotated 171 non-polar and 225 polar metabolites from cured tobacco leaves. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that two growing years and two field locations played primary and secondary roles affecting metabolite profiles, respectively. PCA and Pearson analysis, which used nicotine, 11 other groups of metabolites, two locations, temperatures, and precipitation, revealed that in North Carolina, temperature changes were positively associated with the profiles of sesquiterpenes, diterpenes, and triterpenes, but negatively associated with the profiles of nicotine, organic acids of tricarboxylic acid, and sugars; in addition, precipitation was positively associated with the profiles of triterpenes. In India, temperature was positively associated with the profiles of benzenes and polycyclic aromatic hydrocarbons, but negatively associated with the profiles of amino acids and sugar. Further comparative analysis revealed that nicotine levels were affected by weather conditions, nevertheless, its trend in leaves was independent of two geographical locations and weather changes. All these findings suggested that climate and geographical variation significantly differentiated the tobacco metabolism.}, journal={Frontiers in Plant Science}, publisher={Frontiers Media SA}, author={Ma, Dong-Ming and Gandra, Saiprasad V. S. and Manoharlal, Raman and La Hovary, Christophe and Xie, De-Yu}, year={2019}, month={Oct} }
 @article{la hovary_danehower_ma_reberg-horton_williamson_baerson_burton_2016, title={Phytotoxicity and Benzoxazinone Concentration in Field Grown Cereal Rye (Secale cereale L.)}, volume={2016}, ISSN={["1687-8167"]}, url={https://www.hindawi.com/journals/ija/2016/6463826/}, DOI={10.1155/2016/6463826}, abstractNote={Winter rye (Secale cerealeL.) is used as a cover crop because of the weed suppression potential of its mulch. To gain insight into the more effective use of rye as a cover crop we assessed changes in benzoxazinone (BX) levels in rye shoot tissue over the growing season. Four rye varieties were planted in the fall and samples harvested at intervals the following spring. Two different measures of phytotoxic compound content were taken. Seed germination bioassays were used as an estimate of total phytotoxic potential. Dilutions of shoot extracts were tested using two indicator species to compare the relative toxicity of tissue. In addition, BX (DIBOA, DIBOA-glycoside, and BOA) levels were directly determined using gas chromatography. Results showed that rye tissue harvested in March was the most toxic to indicator species, with toxicity decreasing thereafter. Likewise the BX concentration in rye shoot tissue increased early in the season and then decreased over time. Thus, phytotoxicity measured by bioassay and BX levels measured by GC have a similar but not identical temporal profile. The observed decrease in phytotoxic potential and plant BX levels in rye later in the season appears to correlate with the transition from vegetative to reproductive growth.}, journal={International Journal of Agronomy}, author={La Hovary, C. and Danehower, D. A. and Ma, G. and Reberg-Horton, C. and Williamson, J. D. and Baerson, S. R. and Burton, J. D.}, year={2016} }
 @article{dalal_yalamanchili_hovary_ji_rodriguez-welsh_aslett_ganapathy_grunden_sederoff_qu_et al._2015, title={A novel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for genetic transformation of plants}, volume={81}, ISSN={["1095-9890"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84938634755&partnerID=MN8TOARS}, DOI={10.1016/j.plasmid.2015.06.003}, abstractNote={The rapidly advancing field of plant synthetic biology requires transforming plants with multiple genes. This has sparked a growing interest in flexible plant transformation vectors, which can be used for multi-gene transformations. We have developed a novel binary vector series, named the PC-GW series (GenBank: KP826769-KP826773), for Agrobacterium-mediated plant transformation. The PC-GW vectors use the pCAMBIA vector backbone, and contain NPTII, hpt, bar, mCherry or egfp genes as selectable markers for plant transformation. In a modified multiple cloning site (MCS) of the T-DNA region, we have placed the attR1, attR2 and ccdB sequences for rapid cloning of one to four genes by Gateway™-assisted recombination. In addition, we have introduced four meganuclease sites, and other restriction sites for multi-gene vector construction. Finally, we have placed a CaMV 35S promoter and a 35S terminator on the 5' and 3' ends of the MCS. The CaMV 35S promoter is flanked by PstI restriction sites that can be used to replace it with another promoter sequence if needed. The PC-GW vectors provide choices for selectable markers, cloning methods, and can accommodate up to eight gene constructs in a single T-DNA, thereby significantly reducing the number of transformations or crosses needed to generate multi-transgene expressing plants.}, journal={PLASMID}, author={Dalal, J. and Yalamanchili, R. and Hovary, C. La and Ji, M. and Rodriguez-Welsh, M. and Aslett, D. and Ganapathy, S. and Grunden, A. and Sederoff, Heike and Qu, R. D. and et al.}, year={2015}, month={Sep}, pages={55–62} }